Offprint from "Marine Biology" International Journal on Life in Oceans and Coastal Waters, Vol. 14, No.2, May 1972, Pages 130-142

© by Springer- Verlag 1972 . Printed in Germany

Carbohydrases of some marine invertebrates with notes on their food and on the natural occurrence of the carbohydrates studied

J. HYLLEBERG KRISTENSEN

Zoological Institute, Laboratorium B, Ecology, University of Aarhus; Aarhus, Denmark

130 J. HYLLEBERG KRISTENSEN: Carbohydrases of ma.rine invertebrates 111ar. Bioi.

Abstract

Extracts of 22 marine invertebrates were incubated with 29 different carbobydrates, and enzyme speotra estimated from chromatograms. Enzymatic activities were quantified as reducing sugar in 14 of these species. Significant hydrolysis of laminaran, glycogen, and amylose was found in nearly all species, while hydrolysis of oligosaccharides and structural polysaccharides in most cases was weak or absent. The strongest overall degradation of carbohydrates was found among crustaceans. Enzymatic degradation of laminaran shows some relation to food, but the spectra of carbohydrases are not directly predictable from knowledge of natural food sources. Some carbohydrases (sucmse, cellobiase, lactase) could not be explained by ecological considerations. The results indicate that structural polysaccharides are utilized only to a small extent.

Introduction

FENCREL (1970) observed that bacteria and other microflora were removed from detrital particles during their passage through amphipods, while the detritus itself remained undigested. This observation, and stud­ies by CROSSLEY et al. (1963), GEORGE (1964), NEWELL (1965), HARGRAVE (1970), and KrSSELEvA and VITYUR (1970), indicate that detritus, defined as dead organic matter, plays a minor role in the nutri­tion of detritus feeders.

This paper presents the in vitro activity of carbo­hydrases extracted from benthlc animals mainly ingesting detritus composed of inert substances (Fox, 1950) such as structural carbohydrates. The enzy­matic splitting of cellulose, hemicellulose and algal poly-uronic acids was, therefore, especially studied. An attempt is made to estimate the significance of these substances as energy sources of detritus feeders by comparing the activity of carbohydrases. In addi­tion, some commonly used oligo- and polysaccharides were included in the study to permit comparison with more readily hydrolysed substrates.

An increasing interest in carbohydrases during the last decades has resulted in a number of papers pre­senting different approaches. Comparative studies on degradation of carbohydrates have been carried out on groups of ecologically comparable species. NIELSEN (1962) made a qualitative comparison of

carbohydrases in 34 soil and litter invertebrates, and B. O. NIELSEN (1966) in 6 wrack invertebrates. The present work is, to my knowledge, the first of this kind which compares carbohydrases in marine in­vertebrates, many of which have common feeding habits. Other papers deal with the spectrum of carbo­hydrases of single taxonomical groups or of species; these studies deal chiefly with the quantitative aspect of enzymatic hydrolysis. Degradation has also been investigated in selected animals with respect to particular carbohydrates. Many papers deal with cel­lulases (references by YOKOE and YASUMASU, 1964) chitinases (extensively treated by JENIAUX, 1963), laminaranases (SOVA et a.!., 1970), and alginases (FRANSSEN and JENIAUX, 1965; FAVARO V and VAS­KOVSKY, 1971).

Materials and methods

For the qualitative studies, 19 detritus feeders and omnivorous species were collected during spring and early summer from the estuarine Niva Bay (Muus, 1967), i.e., the bivalves Macoma balthica (L.), Mya arenaria L., Mytilus edulis L., and Cardium edule L., the gastropods Hydrobia ulvae (PENNANT), H. ven­trosa (MONTAGU), and Litto1'ina littorea L., the amphl­pods Gammarus za.ddachi SEXTON, Bathyporeia pilosa LINDSTROM, B. sarsi WATKlN and Corophium volll­tatar (PALLAS), the decapods Carcinus maenas L., a.nd Crangon crangon (FABR.), the isopod ldothea balthica (PALLAS), the barnacle Balanus crenatus BRUG., the polychaetes Nereis divel'sico/{)l' O. F. MULLER, Pygo­spio elegans CLAPAREDE, Arenicola marina L., and the oligochaete Paranais littoralis MULLER. Three species, the gastropod Nassarius reticulatus (L.) the sea star Asterias rllbens L. and the brittle star Ophiacomina nigra ABILDG., were collected in the 0resund and used for comparative purposes. They were fed mussel meat and kept for varying periods of time in aquaria. The other species were dissected shortly after collection, and digestive organs (Fig. 1) were ground at 6°C with HCl-washed quartz. Some small species (Fig. 1) were washed in buffer and homogenized in to/.().

Vol. 14, No.2, 1972 J. HYLLEBERG KRISTENSEN: Carbohydrases of marine invertebrates 131

Samples were diluted to ca. 1: 25 with citric acid­phosphate buffer, pH 5.6. Some extracts were also made at pH 7.6. (Hydrobia ulvae H. ventrosa, Nassa­rius reticulatus and Asterias rubens) and pH 4.3. (N. reticulatus, Ophiocomina nigra and A. ru,bens).

After centrifugation, the supernatant was used as enzyme preparation and 20 fJ.I added to each of the following 29 commercial carbohydrates (about 0.5 mg of each): the <x-glucosides - maltose, sucrose and trehalose (Merck), melezitose (Sigma); the ,a-glucoside cellobiose (Merck); the <x-galactosides raffinose (Merck) melibiose (Sigma); the ,a-galactoside lactose (Merck); the polysaccharides amylose (B.D.H.), laminaran (Nutritional Biochemical Corporation, Cleveland, USA) dextran and xylan (Fluka), glycogen and cellulose (Merck), kappa carrageenan and 2 types of citrus pectin (Copenhagen Pectin Factory), carrageenan, agar-agar, furcellaran, hypnean, iridophycan, Na­alginate and eucheuman (Litex, Copenhagen), chitin and hyaluronic acid (Sigma), alginic acid, gum arabic and methylcellulose (origin unknown).

Incubations were made under a cover of toluene at 32°C for 12 to 16 h. The degree of hydrolysis was estimated with paper chromatography by visual com­parison with control preparations, 1 fJ.l samples were spotted on Whatman No.1 paper and run at room temperature (ca. 22 DC) with n-butanol, glacial acetic acid, and distilled water (4:1:1) as a solvent, and developed according to TREVELYAN et al. (1950).

In the quantitative study, 14 of the mentioned species (Table 1) were collected during spring and early summer along the east coast of Jutland in Kysing Fjord (Muus, 1967) and in Kal0 Vig.

Extracts were made shortly after collection, in distilled water, and filtered by serial filtration through Millipore prefilter AP 25, SS 3 fJ. and HA 0.45 fJ. freeze­dried (Hetosicc CD3) and stored at 5°C. No measurable decrease in activity was found after storage up to 3 months. Immediately before use, the dry powder was dissolved in a phosphate-citrate buffer, pH 5.6 or 7.6. The reaction mixtures contained 0.7 mg extract in 100 fJ.l buffer and 5 mg oligosaccharide or 0.5 mg polysaccharide. The former was added as solution, and the latter weighed on a micro balance (Mettler M5) and added as dry material.

All incubations were made at 32°C under toluene. The first measurement of reducing sugar (RS) was made within 15 min. At least 4 additional samples were analysed at intervals during the following 6 h. The samples were heat-inactivated, deproteinized and RS-measured according to NELSON (1944). After centrifugation, half of the supernatant (1 mI) was diluted to 20 mI and extinction measured at 520 nm (Beckman B). RS was calculated as glucose or N­acetyl-glucosamin equivalents (chitinolysis) after sub­traction of a 9arbohydrate and an enzyme control.

Parallel incubations of identical reaction mixtures gave a standard deviation of ±8.3% (n= 10) in

17'

calculated RS. Incubations were read at constant intervals.

Methods based on alkaline copper tartrate have been commonly used in similar studies, and HARNDEN (1968) reports an accuracy higher than ±1.5% in the range of RS measured by the author. Possibly, the lower accuracy in this study is due to a higher concen­tration of extract used in order to ensure sufficient activity of all enzymes present in crude extracts. Thus, many enzymes were present in concentrations far exceeding the amount of protein, giving direct pro­portionality between enzyme concentration and hy­drolysis. Specific activity was not calculated, therefore, and the absolute RS quantity was used as a measure of carbohydrase activity.

Other methods to estimate RS were tried, but neither colorimetry nor titration according to SoniO­GYI (1945) gave better reproducibility.

Many enzyme preparations decreased the viscosity of pectins, mucoid, and algal colloids, but these hydrolytic reactions were not studied.

Results

<x-glucosides

Maltose

This sugar interferes with liberated glucose and, therefore, hydrolysis was studied only by chromatog­raphy (Fig. 1). Hydrolysis was strong in all incuba­tions at pH 5.6, pH 4.3, and 7.6. Maltase was used as a control of enzyme preparations.

The main digestive function of maltase in vivo is evidently to complete break-down of maltose liberated by digestion of starch, but free maltose may be a minor constituent of the food. Maltose occurred in extracts of a red alga (NAGASHIMA et aI., 1969a) and is ex­creted by certain symbiotic algae (CERNICHIARI et aI., 1969). In contrast, free maltose is common in higher plants (KARRER, 1958). In animals, maltose is accu­mulated in a crayfish (SPECK, 1969).

Sucrose

In the qualitative study, medium or strong hydrol­ysis was found in most of the species (Fig. 1). The quantitative study showed low activity in Balanus crenatus and a still weaker activity in Arenicola marina. As a rule, crustaceans showed the strongest activity, e.g. ldothea balthica and Oorophium Volu­tator (Table 1). This relatively strong sucrose-splitting is remarkable, as there is no indication of a higher level of sucrose in their food.

The pH of 5.6 is close to the optimum value found by HORIUCIII (1963) in a marine bivalve. KOOIMAN (1964) reports that the pH optima for both maltase and sucrase ranged between pH 5.0 and 6.0. Sucrose

132 J. HYLLEBERG KRISTENSEN : Carbohydrases of marine invertebrate. Mar. Bioi.

Table 1. A mounts 0/ RS (reducing sugar) liberated alter 1 h, when caku1ated as % 0/ incubated carbohydrate. The amounts are read from curves as shown in Fig. 2, which atso indicates reaction mixtures and condition

0/ incubation. T: less than 0.01 % RS, H: hepatopancreas: I: intestine: W: whole organisms

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Sucrose 0.6 1.0 0.6 1.1 0.6 Melezitose 0.8 1.8 0.8 0.4 0.2 Trehalose 0.3 1.0 1.1 0.2 0.4 Raffinose 0.3 0.2 0.4 0.7 0.2 Amylose 11 4 10 21 13 Glycogen 21 13 24 40 24 Laminaran 47 58 60 70 40 Dextran 0.6 3.2 0 2.0 0 Cellulose 0.1 2.0 0.1 0 1.0 Methylcellulose 2.4 5.0 2.4 4.0 1.6 Xylan 0.1 2.8 1.0 0.4 2.4 Aginic acid 0.8 2.0 0.2 0 3.2 Na·alginate 0.4 1.2 1.0 0 1.2 FurceIJaran 0 0.8 1.2 0 0 Chitin 6 10 3 6 7

is a metabolic product of all chlorophyll. containing plants (KAB.:aER, 1958), often abundant in vascular plants and recorded from several green, red and brown algae (PERCIVAL and DOWELL, 1967), although in low concentrations (LEVRING et aI., 1969; NAGASHIMA et al. 1969b). Observations on free sucrose in animals are rare (WYATT, 1967).

Melezitose

This trisaccharide is composed of 2 glucose and 1 fructose molecules. It seems to contain both an IX­

and a p-linkage, but the chemical structure is not completely known. In the qualitative study, signifi­cant hydrolysis was found only in the 4 bivalves. Limited evidence of degradation was found in some other species (Fig. 1) and, at pH 4.3, some hydrolysis occurred in Ophiocomina nigra extracts.

The quantitative study (Table 1) showed that extracts other than those mentioned hydrolyse melezi­tose, but that the activity is weak. The RS values often culminated after 1 to 2 h, after which RS decreased. This may explain the differences between the two methods, as the qualitative preparations were incubated for 12 to 16 h.

Melezitose occurs in the cell sap and excretions of certain higher plants (KARRER, 1958), and there are no reports on melezitose in marine algae or other potential food sources of marine animals.

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Trehalose

Like maltose, this sugar is composed of 2 glucose molecules, but they are coupled via an IX - IX bond. The qualitative study showed degradation at pH 5.6 in the 4 bivalves, and in Gammarus zadr1achi, Grangon crangon, Gorophium vol;utator and ldothea balthica. At pH 7.6, trehalose was also hydrolysed by Hydrobia ventrosa, H. ulvae and Littorina littorea, suggesting that the pH optimum may be near the neutral point.

In the quantitative study, trehalose was split by all species, although never exceeding 1 % degradation after 1 h. Bivalves showed the strongest activity, while in Littorina littorea and Arenicola marina, activi­ty was almost negligible.

Trehalose is widely distributed. It is sparsely found among higher plants, but abundant in algae, fungi and bacteria (KARRER, 1958). Trehalose is the most im­portant oligosaccharide in insects and has also been identified, sometimes in high concentrations, in other invertebrates (GILMOUR, 1961; SCHWOCH, 1969; SPECK,

1969).

p-glucosides

Cellobiose

This sugar is reducing, and has been tested only qualitatively. At pH 5.6, cellobiose was hydrolysed by extracts of 17 of the species tested (Fig. 1).

Vol. 14, No.2, 1972 J. HYLLEllERG KRISTENSEN: Carbohydrases of marine invertebrates 133

weak to strong • hydrolysis (RS)

very weak RS record no hydrolysis

not tested also quantita ~ o tive evidence of hydrolysis

Maltose Sucrose Melezitose Trehalose Cellobiose Raffinose Meli biose Lactose Amylose Glycogen Laminaran Dextran Methylcellulose Cellulose Chitin Xylan Standard Pectin Pure Pectin Gum arabic Alginic acid

pH 7.6

Na-alginate pH-7.6

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Fig. 1. Qualitative enzyme spectra. In the 14 species also treated quantitatively, encircled dots show RS (reducing sugar) value of more than 16 [Lg RS after 1 h. Lower values are a.rbitrarily termed very weak. C: orystalline style; D: diver­tic\es of amphipods and oesophagus divertic\es of Arenicola; H: hepatopanoreas; S: stomach; I: intestine; SF: stomach fluid; W: whole organisms. Vertical lines separate taxonomical groups; horizontal line separates oligo- and polysaccharides

Cellobiose is not accumulated as a free sugar, but used by plants in the biosynthesis of other carbohy­drates (KARRER, 1958). Generally, the occurrence of cellobiose is ascribed to enzymatic degradation of cellulose and lichenin by bacteria and fungi. WIN­

TERINGHAM (1965) mentions one single record of free cellobiose in insects.

O<.-galactosides Raffinose

In the qualitative study, hydrolysis of this tri­saccharide could only be demonstrated in the 4 bi­valves, apart from a very weak reaction in Grangon crangon. The few experiments at pH 4.3 an 7.6

failed to demonstrate hydrolysis. The quantitative study, however, gave evidence of very weak hydrolysis in other species too (Table 1).

Raffinose is well known from sugar beets, but is also co=on in other vascular plants (KARRER,

1958; WANNER, 1958). Its occurrence in algae is doubtful. According to LOVERN (1958), an isomer of raffinose is reported from bacteria, but apart from this evidence raffinose seems to be sparsely found, except in higher plants.

Melibiose This disaccharide was studied only qualitatively.

Hydrolysis occurred at pH 5.6 in 7 species (Fig. 1).

134 J. HYLLEBERG KRISTENSEN: Carbohydrases of marine invertebrates Mar. Biol.

In nature, it is restricted in occurrence to secre­tions, leaves and fruits of higher plants.

{J -galactoside.s

Lactose

Like melibiose, lactose gives galactose and glucose on hydrolysis, but the 2 molecules are coupled via a {J-linkage. The sugar is reducing, and was only studied by chromatography. At pH 5.6, it was hydrolysed by 15 species (Fig. 1). At pH 4.3, hydrolysis was evident in Ophiocornina nigra. At the same pH, extracts of N Msarius reticulatus and AsteriM rubens also showed lactolytic activity. At pH 7.6, Littorina littona extracts hydrolysed lactose.

Milk of mammals is the only lactose source known in animals, but it has occasionally been isolated in higher plants (KARRER, 1958).

Polysaccharides, re.serve carbohydra,te.s

Amylose

This 1,4-linked (X-glucan was well hydrolysed at pH 5.6 by all extracts except from Ophiocornina nigra, where only weak activity could be demonstrated.

Animal amylases belong to the (X type, giving oligosaccharides (finally maltose) at hydrolysis. A step-wise hydrolysis could be observed on the chroma­tograms of all species, except the echinoderms Aste­riM rubens and Ophiocom.ina nigra, where only glucose spots were identified.

The quantitative study showed a variation in RS-liberation after 1 h, ran"oing from 33 % in ldothea balthica to 4% in Mytilus edulis (Table 1). AGRAWAL (1963) found an amylase optimum near pH 5.8 in Corophium volutator, and BLANDAMEER and BEECHEY (1964) an optimum at 7.0 in CMcinus m.aena8. I found an optimum near 6.2 in M. edulis, but a pH of 5.6 was retained throughout, as the pH effect itself appeared to be small. This finding can be compared with observations by BLANDAMEER and BEECHEY (1964), who found that the specific activity in hepatopancreas extracts of C. m.aenM rose from 1.3 fJ. mol maltose/min/ mg protein at pH 5.6 to 1.8 at the pH 7.0 optimum (figures taken from their Fig. 1).

Amylose is a reserve carbohydrate of all chloro­phyll-containing plants. Probably all green algae con­tain starch similar to that of land plants, comprising both amylopectin and amylose (PERCIVAL and Dow­ELL, 1967). The starch of Rhodophycea (floridean starch) and of Cyanophycea also contain an (X-l,4-linked glucan (MEEUSE, 1962; PERCIVAL, 1968), while the starch of Phaeophycea consists of the i'i-l,3-linked glucan laminaran.

Many forms of bacteria produce starch-like glucans identical in structure to those produced by yeast and higher plants (FOSTER and STACEY, 1958).

Glycogen

In the qualitative study, this 1.4 and 1.6-linked (X-glucan was split by all species, but hydrolysis in Ophiocom.ina nigra was weak compared with the other species.

The quantitative study showed hydrolysis of gly­cogen, which generally followed the pattern of amylase activity. In 11 species, the ratio of glycogen to amylose (G/A) hydrolysis was about 2: 1. In Mytilus edulis, the ratio was nearly 3: 1, and in Balanus CTenatus and AsteriM Tubens, about 1: 1. This result can be compared with the finding of FmQUET and DANDRIFOSSE (1967), who studied G/A ratios in vertebrates and found them to be about 1: 2 in herbivores and 1: 1 in carnivores.

The G/A ratios computed from the present data appear to be independent of the nutritional type of the species investigated.

Amylase is known to act on glycogen but, according to WYATT (1967), a further enzyme is required for glucose production. MARZLUFF (1969) found that (X-glucosidase, in casu sucrase might fulfill this ex­pected role. The present study gives little evidence of such correlation.

Glycogen is similar in structure to amylopectin from plants, but the molecule is more branched and contains more glucose units (KARLSON, 1962). Gly­cogens from bacteria are similar to those of animal origin (FOSTER and STACEY, 1958; ANTOINE and TEpPER, 1969).

Dextran

This glucan contains a high proportion of (X-1,6-glucosidic linkages. At pH 5.6, the chromatograms had distinct spots in A1·enicola marina, Balanus crenatus and Corophium volutator, while only weak hydrolysis could be observed in 6 other species.

The quantitative study revealed the highest RS value in Arenicola 1narina, although only 4 % RS was liberated after 1 h (Table 1).

A specificity of the dextran-splitting enzyme can­not be estimated from my investigation, but BRUNI et al. (1969). found that a highly purified acid (X-gluco­sidase hydrolysed glycogen, maltose and dextran at pH 4.5

Dextran is a constituent of certain chain-forming bacteria (FOSTER and STACEY, 1958). It is likely to be rare in marine environments.

Laminaran

In the qualitative study, the {J-1,3-linked glucan laminaran was significantly split by all species except AsteriM 1·ubens. A weak hydrolysis was found in ex­tracts tested at pH 4.3 and 7.6. The laminaran was split into glucose and laminamn dextrins, mainly laminarabiose.

Laminaran was found to be efficiently split by Compkium volutato-r (80% RS after 1 h), while

Vol. 14, No.2, 1972 J. HYLLEBERG KRISTENSEN: Carbohydrases of marine invertebrates 135

JIg AS

400 • Laminaran

•

200 Glycogen o

o

Amylose

+ +

+ Dextran

2 3

Fig. 2. Arenicola marina. 0.7 mg extract; 0.5 mg polysaccha­ride; 100 V-l buffer pH 5.6; 32°C. Degradation of reserve carbohydrates by extracts of intestine. RS (reducing sugar)

values are calculated as glucose equivalents

AGRAWAL (1963) failed to demonstrate this in tests with the same species. Asterias l'ubens yielded 3 %, and Cal'cinus maenas 14% RS, and the other species be­tween 40 and 70% after 1 h (Table 1).

The present findings can be compared with the observations by STONE and MORTON (1958), although there are slight differences regarding su bstra tes (soluble laminaran contra insoluble laminaran) and handling of the extracts. STONE and MORTON re­corded a stronger activity in Cardium edule and Mya arenaria than in Littorina littorea and Nassarius reticulatus, which agrees with my own observations.

The comprehensive study on laminaranase by SOYA et al. (1970) cannot be used for direct com­parison with the present investigation, as only one species is common to both works. The general impres­sion given by the data of SOYA et al. is a strong activity in crustaceans, and a somewhat lower in hepatopancreas extracts of molluscs, while echinoderms have even lower values. The same pattern is evident in the present study. .

The pH of 5.6 is near the optimum value (SOYA et al. (1970). NIELSEN (1963) reports optima in two terrestrial invertebrates at pH's of about 5.0 and 5.5, respectively.

GJucans of the same chemical type as laminaran are probably the most common polysaccharides in nature (MEEUSE, 1962). The J'i-1,3-linked glucan applied in this investigation was extracted from a brown alga (Laminaria sp.). The polysaccharide is a common storage product of most Phaeophyta. In addition, J'i-1,3-Jinked glucan is reported from Crysophyta, diatoms (PERCIVAL, 1968) and euglenoids (LEEDALE, 1967). According to HANDA (1969), glucan comprised a main component of the marine particulate matter studied by him. Glucan also occurs in callose and cell walls of plants (MEEUSE, 1962).

Structural polysaccharides

Cellulose

Hydrolysis of this J'i-1,4-Jinked glucan was, in the qualitative study, only detected in extracts of the crystalline style of Mytilus edulis and of the hepato­pancreas of Littorina littorea, but cellulolysis was weak compared with the other carbohydrases investig!Lted.

The quantitative analysis indicated hydrolysis by Corophium volutator of about the same order of magnitude as in Mytilus edulis, i.e., about 2% RS liberated after 1 h. The other species investigated ranged from 0 to 1 % RS after 1 h (Table 1).

GJucans of the cellulose type are widely distributed in vascular plants, and are present in most algae (PERCIVAL, 1968), fungi, and also certain bacteria. This type is rare in .. animals, but occurs in tunicates.

Methylcellulose

This polymer is obtained by exchange of -CHa with -OH groups in cellulose, and gives a water­soluble cellulose-derivate.

At pH 5.6, weak chromatographic evidence of hydrolysis was found in .Macoma balthica, Mytilus edulis, Cardium edule, and Hydrobia ulvae and H. ven­trosa; therefore, decomposition of methyl cellulose seems to be somewhat easier than that of crystalline cellulose, as confirmed by quantitative studies (Table 1). It has been shown recently that C. edule has cellulase attacking degraded but not crystalline cellulose (KOOPMANS, 1970). This was confirmed by my own observations.

Chitin

This polymer is composed of J'i-1,4-linked N-acetyl­glucosamine units.

The chitin studied was prepared from finely powdered crab shells. It was hydrolysed at pH 5.6 by all extracts, except the snails Hydrobia ulvae and H. ventrosa, where hydrolysis occurred at pH 7.6. In extracts of other species, chitinase activity was ob­served at pH 7.6 and 4.3.

Quantitative measurements showed a higher liber­ation of RS in crustaceans, while Arenicola marina

136 J. HYLLE1lERG KRISTENSEN: Carbohydrases of marine invertebrates Mar. BiOI.

had the lowest activity (Table 1). The estimated chitin­nolysis comprises the activities both of chitinase and chitobiase calculated as N-acetylglucosamin equi­valents. The reducing capability of N-acetylglucos­amine is only about i of that of glucose.

Chitin is a structural component in several in­vertebrate phyla, especially arthropods, molluscs and annelids (JENIAUX, 1963). The polymer is also known to occur in fungi and certain algae, e.g. diatoms (McLACHLAN et aI., 1965).

Xylan

This pentosan is principally a ,B-1,4.linked polymer of xylose, but also 1,3-linkages occur. Only one case of xylanase activity was observed in the qualitative incubatious. A spot with an R j value corresponding to xylose indicated degradation of xylan by ldothea balthica. The chromatographic records were not in accordance with the RS measurements, where all extracts indicated hydrolysis of xylose ranging from 4% RS in Littorina littorea to 0.1 % in Arenicola marina after 1 h (Table 1).

Xylan is widely distributed in cell walls of vascular plants. Smaller amounts of other compounds are usually found associated with xylans, their nature depending on the origin of the polymer. There a,re several reports on xylans in marine algae (KREGER, 1962), especially in Rhodophyta (PERCIVAL and Dow­ELL,1967).

Pectin

The constituants, i.e., sugars, uronic acids, the degree of estrification and percentage of insoluble protopectin, varies according to the origin of the pectin, but the polymer is principally a chain of iX-1,4-linked galacturonic-acid units.

Hydrolysis of pectin was only tested by ohromatog­raphy. The high-ester pectin (estrificated in 67.6% of the carboxyl groups) was not hydrolysed in any of the incubations. An unspecified standard pectin tested had spots on the chromatograms with an R j value corre­sponding to glucose in about half of the incubations. In this case, however, it is obvious that the spots are due to hydrolysis of oligosaccharides present in the standard pectin.

Pectins are widely distributed, but found mainly in vascular plants.

Gum arabic

This acidic polysaccharide occurs in Acacia species. It contains several sugars and uronic acids (HIRST and JONES, 1958), and varies in composition depending on origin.

Incubation with gum arabic did not reveal reducing carbohydrates in the qualitative tests, and quantitative studies were not carried out.

Alginic acid and alginate

These polysaccharides are polymers of mannuronic and guluronic acids connected by 1,4 and possible also 1,3 linkages. Sodium alginate is a water-soluble salt of alginic acid, but varies in uronic-acid proportions as well as accompanying sugars.

In the qualitative analysis, degradation of alginic acid was not observed in the species tested at pH 5.6, but it was split at pH 7.6 by Littorina littorea and, to a lesser degree, by Hydrobia ventrosa. The sodium salt was split by the same species both at pH 5,6 and 7.6. At pH 7.6, weak hydrolysis of Na-alginate was observed in NassariU8 nticulatU8.

Measurements at pH 7.6 gave a higher liberation of RS from alginic acid than from Na-alginate (Table 1). The pH 7.6 is close to the optimal values recorded by FRANSSEN and JENIAUX (1965).

A polysaccharide similar to alginic acid has been isolated from bacteria grown under artificial conditions but, apart from this record, the occurrence of this polysaccharide seems to be restricted to brown algae (PERCIVAL and DOWELL, 1967).

Hyaluronic acid

This polymer consists of glucuronic acid and N­acetylglucosamine connected via 1,3-linkages to chain­formed macromolecules.

Degradation of hyaluronic acid was tested only by chromatography. The viscosity of the mucoid was obviously decreased by many of the extracts, but complete degradation never occurred, and oligogly­cosides were never observed. KITAMIKADO and YA· MAMOTO (1969, 1970) investigated hyaluronidase activity in fish, some invertebrates and bacteria, and found chromatographic evidence of di- and tetra­saccharides as products of hydrolysis. The pH optima of enzymes of fish and bacteria were 3.5 and 6.2, respectively.

Hyaluronic acid comprises a significant part of connective tissue and many slimy secretions in ani­mals.

Mucilage-like polysaccharides

None of the polysaccharides, carrageenan, kappa carrageenan, hypnean, eucheuman and iridophycan, liberated RS except to a small extent in Asterias rubens (Fig. 1). An unidentified reducing-sugar (Rj

close to glucose) was found in agar incubations in some of the species at pH 5.6 (Fig. 1). Furcellaran was negative on all chromatograms, but incubations in­vestigated photometrically showed an increases in RS from 0.1 to 1,8% after 1 h at pH 7.6 (Table 1).

The polysaccharides are characterized by linkage of sulfate esters with a galactan, although several sugars other than galactose have been identified in hydrolysates.

Vol. 14, No.2, 1972 J. HYLLEBERG KRISTENSEN: Carbohydrases of marine invertebrates 137

Carrageenan is a crude extract of the red algae Chondrus stellata. Kappa carrageenan is the residue in the supernatant after HCl extraction of carrageenan.

Extracts from the red algae Hypnea and Iridea are termed hypnean and iridophycan, respectively, and are similar in composition to carrageenan.

Agar-agar is extracted from red algae of the genera Gelidium and Gracillaria. Furcellaran (Danish agar) is a 1,3-linked galactoglycane sulphate isolated from the red alga Furcellaria jastigiata. Eucheuman is extracted from Eucheuman cottonii.

Discussion

Evaluation of the metabolic significance of enzymes from crude extracts is not easy since their activity can be interpreted in different ways. Firstly, enzymes play an important role in digestion when acting on readily hydrolysable material. Secondly, certain enzymes may act as a "tool" to open cell walls and make hydro­lysable matter more readily accessible. Thirdly, en­zymes may be produced by microorganisms in the gut of the animals studied.

The methods of the present work should cover all three possibilities. However, the following discussion is based chiefly on the first-mentioned possibility, as a relatively strong hydrolysis is likely to occur in carbo­hydrates with a potential food value. Enzymes with "tool" function may be difficult to evaluate, but it is possible that some of the enzymatic activities termed "very weak RS records" in Fig. 1 belong to this category. Enzymes such as cellulase may be important in the digestive process, even when present in small quantities.

As regards the enzymes of microorganisms in the gut, it is evident that these enzymes cannot be distinguished from the host enzymes by the tech­nique used here. However, bacterial enzymes are be­lieved to occur only in small quantities in the extracts, as there are no morphological adaptations of the gut of the animals studied which would enable quantities of microorganisms to be harboured.

It has often been discussed whether enzyme spectra may provide information on the food sources of animals or not. According to WEEL (1961), the general opinion is that enzyme equipment and secretion "fit" the diet. An evaluation of the enzyme­food relation from the present study seems to depend on whether the carbohydrases are treated "one by one" or on whether the total number of significantly hydrolysed carbohydrates is considered. A one-by-one analysis only gives limited information in this respect. This conclusion coincides with the result of NIELSEN (1962), who found a poor - if any - correlation be­tween the type of diet and the carbohydrases present in the alimentary tract of soil and litter invertebrates.

This poor correlation can be explained by two facts: (1.) A number of carbohydrates are of universal

18 Marine Biology. Vol. 14

occurrence in plants, and may be found in animals also. In other words, demonstration of, e.g. trehalase activity in crude extracts, cannot show whether the tested animals posses the trehalase to digest plants or other animals, or whether the enzyme is used else­where in the metabolism of these species; (2) according to earlier theories (VEIBEL, 1958), only 5 different en­zymes are necessary to hydrolyse all glycosides. Nei· ther this. theory nor the view that hydrolysis of each glycoside demands a specific enzyme are accepted today. There are many records of highly purified enzymes attacking a number of substrates with identical linkage between the molecules (cf. BRUNI et aI., 1969; HASEGAWA and NORDIN, 1969).

Enzymes hydrolysing carbohydrates which are unlikely to occur in natural food may be considered from this view point. If a specific cellobiase exists (VEIBEL, 1958; OKADA et al., 1966), the strong cello­biase activity found in most of the investigated species is amazing, as cellobiose is only known to occur free in the last step of the decomposition of cellulose ..

NIELSEN (1962), PENTREATH (1969), and TELFORD (1970) also observed that cellobiose was strongly hydrolysed, even when degradation of cellulose could not be demonstrated by the sll-me extracts. The natural substrate is likely to have universal occurrence, since cellobiase has been demonstrated in terrestrial, limnic, and marine invertebrates. Possible compounds would be glucons connected via {i-linkage to an aglu­con. Amygdalin is a {i-glucoside of this type, and is hydrolysed by the {i-glucosidase emulsin, which also acts on cellobiose.

The glucon of amygdalin is gentiobiose, which has the glucose molecules· connected via {i-1,6-linkages, indicating that the con£guration at - 0 - is more important for the action of the enzyme than the link­age number. Emulsin is a typical plant enzyme and, according to GOTTSCHALK (1958), it has been established that plant {i-glucosidases from different sources vary in specificity. Some act on cellobiose, gentiobiose and other {i-D-glycosides, while others have more restricted activity.

JENIAUX and DEVIGNE (1960) observed that com­mercial {i-glucosidase hydrolyses chitobiose, and this ought to be considered in future studies on the natural function of cellobiase. Chitobiose consists of two N­acetylglucosamine molecules linked by a {i-1,4-linkage, and it is split by chitobiase which, by some authors, is considered identical with N-acetylglucosaminidase (JENIAUX, 1963). The latter enzyme is common in digestive tissues, for example in various granules in the hepatopancreas of molluscs (SUMNER, 1969). It is known that digestive cell extracts of Mytilus cali­jornianus posses lytic activity dissolving bacteria rapidly (ZOBELL and LANDON, 1937). Cell walls of bacteria contain peptidoglycan (TIPPER et a!., 1967); lysozyme, (muramidase) shows N-acetylglucosamini­dase activity towards chitin, various saccharides, and

138 J. HYLLEBERG KRISTENSEN: Carbohydrases of marine invertebrates Mar. Bioi.

synthetic substrates, and it acts on bacteria ce11-walls, cleaving the glycosidic bond of the N-acetyl­muramic acid (W ADSTROM and HISATSUNE, 1970). My suggestion is, therefore, that hydrolysis of cellobiose may be caused by lysozyme.

The strong p-galactosidase activity (lactase) of most of the species tested is also interesting. Here, as in cellobiose, hydrolysis is conspicuous, but the occur­rence of lactose in the natural diet is not known. It occurs in milk and in pollen of certain flowering plants. It is likely that lactase, if it occurs in marine inverte­brates, acts on p-galactosides as it does in yeast, bac­teria etc. (GOTTSCHALK, 1958).

Other substrates could be galactolipids of algae, which according to BENSON and SHIBUYA (1962) can be enzymatically degraded by IX- and p-galactosi­dases.

The slight hydrolysis of alginic acid and alginate by the carnivores Asterias rubens and Nassarius reticulatus is another puzzling example (Table 1), as alginic acid is restricted in occurrence to brown algae which are not a food source of these two species. FRANSSEN and JENIAUX (1965) found a hepatopan­creas alginase activity of 1.2 A.U. (alginolytic units) in N. Teticulatus compared with 72 A.U. per g tissue of Littorina littorea (herbivore). In L. littorea, alginic acid may have a nutritional importance or the enzyme is here a tool to open up ingested cell walls. How­ever, in carnivores, the importance of alginase is not known. The enzyme is either present as a reminiscence of ancestral functions, or it attacks a chemical bond which occurs in natural food as well as in alginic acid. Algal polysaccharides similar in structure to animal polysaccharides exist (PERCIVAL and DOWELL, 1967).

The highest number of carbohydrases was found in detritus feeders and microherbivores (bivalves) and the lowest in the presumably carnivorous Ophiocomina nigm. In my opinion, the total number of carbohy­drases should permit a rough estimation of the natural food source of a species, but there is no evidence of a sharp classification between herbivores, carnivores, omnivores and detritus feeders on the basis of different carbohydrase spectra.

Fig. 1. shows that bivalves are able to degrade most glucosides, galactosides and a number of poly­saccharides. Macoma balthica (deposit feeder) ingests the top layer of the sediment (NEWELL, 1965). Mya arenaria, Cardium edule and M ytilus edulis feed on suspended material. In all 4 species, diatoms and other minute plant material are frequently found in the intestines (BLEGVAD, 1914). No doubt exists that plant material is also digested as demonstrated in several feeding experiments, e.g. by WERNSTEDT (1942). WERNSTEDT fed Macoma balthica and Cardium edule with detritus and diatom cultures, and found natural detritus rich in bacteria to be more favourable for growth in M. balthica than in C. edule. With

diatoms, he observed a reverse result. The quantita­tive data on carbohydrases (Table 1) indicate a more effective degradation of carbohydrates by C. edule than by M. balthica. The stronger activity in the for­mer species make my carbohydrase results comparable to the experiments of WERNSTEDT. A more effective degradation of laminaran in C. edule might be ex­pected, since lamiDaran is an important reserve in many diatoms (MEEUSE, 1962).

Some of the crustaceans hydrolysed several carbo­hydrates. The omnivorous ldothea balthica degraded 10 different carbohydrates. This species readily ingests filamentous algae (RAVANKO, 1969), but appears to utilize animal food better (SOLDATOVA et aI., 1969). The qualitative spectra and the liberation of RS were found to be much the same as in Cmngon cmngon (omnivore, BLEGVAD, 1914) and Corophium volutator, a selective detritus feeder which sorts-out diatoms (THAMDRUP, 1935) and various microorganisms from mucous films on sand grains (MEADOWS, 1964). In contrast, Carcinus maenas, an omnivore with prefer­ence for animal food (ROPES, 1968), has a similar spec­trum (11 carbohydrases) but a much lower RS libera­tion (Table 1). A comparison of RS liberation in the four crustaceans, therefore, indicates a different nu­tritive value of carbohydrates. Again, lamiDaran degradation seems to give good evidence for a food and enzyme relationship. LamiDaran may be significant in the nutrition of C. volutator, less so in that of I. balthica and C. crangon and of no significance in C. maenas.

The narrow spectrum of carbohydrates degraded by Bathyporeia pilosa and B. sarsi is remarkable, as they are known exclusively to ingest matter adhering to sand grains (NICOLAISEN and KANNEWORFF, 1969) covered by diatoms, blue-green algae and bacteria (ANDERSON and MEADOWS, 1968, 1969). This low number of carbohydrases is perhaps due to a special­ized diet, since these amphipods live in a special environment of well-sorted sand.

The lowest number of carbohydrases in crustaceans was found in Balanus crenatus. HARNDEN (1968) investigated the carbohydrases of B. nubilis (27 carbohydrates tested), and found a low number of hydrolytic enzymes, viz. maltase, lamiDaranase, chitin­ase, and glycogenase active at pH 5.5 These carbohy­drases were also found to be significantly active in the present study on B. crenatus. The small RS liberation (apart from lamiDaranase) may indicate that zooplank­ton is a major food source of sessile barnacles. BARNES (1969) found diatoms and sand grains only occasionally in the stomach of this species. Experi­ments by BOOKHOUT and COSTLOW (1959) have shown that animal food alone or in combination with Chlamy­domonas support growth of older barnacles to a greater extent than unialgal cultures alone, indicating that, even if the algae are ingested, only a minor part of the carbohydrates are digested.

Vol. 14, No.2, 1972 J. HYLLEBERG KRISTENSEN: CarbohydraJJeS of marine invertebrates 139

Four gastropods were investigated, but the rela­tionship between carbohydrases and diet is not evi­dent. Littorina littorea, an omnivore (BLEGVAD, 1914) which feeds mostly on fucoids (OWEN, 1966), possesses the widest spectrum (10 carbohydrases). However, Nassarius reticulatus is capable of hydrolysing nearly the same carbohydrates, although quantitative differ­ences were obvious. The intestine of N. reticulatus is often filled with bottom material containing many diatoms (BLEGVAD, 1914). This material may orginate from ingested food (bivalves, crustaceans), but the carbohydrases indicate that plant material is also perhaps digested by N. reticulatus. The Hydrobia species ingest bottom material and mucous film scrap­ed off with the radula (NEWELL, 1965). The quanti­tative spectrum (Table 1) is not only very similar to that of the detritus feeding M acoma balthica, but also to the spectrum of N. reticulatus.

Arenicola marina hydrolysed 8 carbohydrates. The small detritus feeding Pygospio elegans hydrolysed 6, but the spectrum is not complete. Nereis diversicolor hydrolysed 8 and the oligochaete Paranais littoralis at least 7 carbohydrates. A comparison of food and enzyme spectra, respectively, is not possible in this group due to lack of data, except in A. marina and N. diversicolor. The former species ingests sand to­gether with filtered particles (KRUGER, 1971), while the latter is omnivorous (GOERKE, 1971) with a pref­erence for animal food (KONIG, 1964, p. 220).

The poor RS liberation obtained with Arenicola marina . extracts indicates that plant material other than diatoms and bacteria is of minor im­portance in the nutrition. This conclusion coincides with experiments by HOBSON (1967), who found that little of the available organic carbon in the sediment was utilized in two North American Arenicola species.

For comparative reasons, two echinoderms collect­ed at deeper water were included in the study, as they are both known to take animal food. The sea-star Asterias 1"ubens hydrolysed 6 carbohydrates, and the brittle star Ophiocomina nigra only 4.

Asterias rubens preys on small bivalves, crusta­ceans etc., and detritus in the stomach (BLEGVAD, 1914) may, as in Nassarius reticula/us, originate from ingested prey. Here again, part of the carbohydrate may be utilized by the predator, although RS measure­ments indicate low efficiency only.

Ophiocomina nigra had the lowest number of carbo­hydrases of the species investigated. However, the spectrum would doubtless be extended if RS measure­ments were carried out. The poor hydrolytic activity (Fig. 1) is surprising, as the brittle star is considered to be omnivorous (REESE, 1966). VEVERS (1956) found that O. nigra occasionally browses on algae. According to FONTAINE (1965), the ophiuroid is capable of ex­tracting plankton by filter feeding, while ROUSHDY and HANSEN (1960) did not find evidence of filter feeding in this species. At any rate, the spectrum of

carbohydrases may call attention to the possibility that O. nigra mainly digests animal food. The signifi­cance of animal food is easily demonstrated in aquaria, where mussel meat stimulates the feeding activity of O. nigra violently.

It appears from the present investigation that carbohydrases bear some relation to the food and trophical classification of marine invertebrates. How­ever, oligosaccharides and reserve carbohydrates of plants may be of nutritive significance in most spe­cies, while utilization of structural carbohydrate is obviously poor, with the few exceptions of alginic acid and alginate. This conclusion is in agreement with other investigations. HUANG and GIESE (1958) investigated some marine herbivores, and found that only readily assimilable material such as reserve carbohydrates were digested. In contrast, BOOLOO­TIAN and LASKER (1964) reported 80% of the dry matter of the brown alga Macrocystis to be removed by digestion in vivo in the sea urchin Strongylocentrotus pU1·puratus. However, they found other algae digested with lower efficiences by the same species (44 to 62%).

The present study also seems to fit available ex­perimental data on the utilization of dead organic material by detritus feeders. NEWELL (1965) suggests that bacteria are an important food source for Macoma balthica. The detritus per se (dead material) only served as a substrate of attached flora. Experiments of FEN­CHEL (1970) on a marine detritus-feeding amphipod, and by HARGRAVE (1970) on a freshwater amphipod, gave similar evidence. FENCHEL (1970) found that detritus particles were completely free from attached Inicroflora (bacteria) during the passage through the alimentary tract of the amphipod. CROSSLEY et a1. (1963) state that terrestrial Inicroarthropods only fragment detritus, but that bacteria and fungi are responsible for the actual decay.

Enzymes hydrolysing cellulose, polyuronic acids and mucilage-like substances are co=only reported in marine invertebrates. However, it appears to be a general characteristic of enzymes acting on cellulose that they need a long exposure time of the substrate for complete digestion, and such long exposure is not likely in the detritus-feeding animals studied. KRU­GER (1971) mentions 14 min as an average time neces­sary for Arenicola marina to renew its intestinal contents. I have measured the renewal time in the deposit-feeding bivalve Abra alba S. WOOD by fecal­pellet countings, and have found that the gut content was renewed in 0.3 g animals in 1 h at 18°C. In other words, it is believed that structural carbohydrates are not degraded during passage through the gut of these animals.

A number of works on cellulases have been pub­lished and, according to Y OKOE and YASUMASU (1964), cellulase is a widely distributed enzyme among invertebrates. Cellulases which may be significant in metabolism have been shown in wood-ingesting spe-

140 J. HYLLEBERG KRISTENSEN: Carbohydrases of marine invertebrates MM. Bioi.

cies such as Teredo (GREENFmLD, 1959), Bankia indica (NAIR, 1955) and Limnoria (several references in ARVY, 1969). However, much work on degradation of carbohydrates is based on viscosimetry or turbidity readings, and great variety in applied substrates may, in addition, make comparisons impossible. The diffi­culties are demonstrated by EpPLEY and LAsKER

(1959), who found viscosimetric evidence of alginase in St·rongylocentrotus purpura/us, butfailed to show RS as a result of the alginolysis.

It is emphasized that the significance of carbohy­drases in the metabolism and turnover of structural elements is, on the whole, unknown in marine in­vertebrates, although there are several reports on enzymatic degradation of these compounds (cf. MEEUSE and FLUEGEL, 1958; GALLI and GIESE, 1959 ; HORIUcm, 1963). However, it can be assumed that bulk degradation of structural polysaccharides will not normally occur through digestive enzymes pro­duced by the herbivores themselves, but possibly in some cases through associated microorganisms. In general, a herbivore will ingest large amounts of plants but only digest a fraction; an omnivore will prefer animal to plant food; and detritus feeders will pass quantities of detritus through the alimentary tract in order to secure the nessessary intake of easily assimilable matter.

Summary

1. Hydrolysis of oligosaccharides was mostly found to be weak in extracts of the species studied.

2. The reserve carbohydrates amylose, glycogen and laminaran were significantly split in nearly all species. Dextran was poorly, if at all, split.

3. Hydrolysis of structural carbohydrates was very poor, with few exceptions formed by methycellulose and alginic acid. An exception was chitinolysis, which occurred in all species.

4. The spectrum of the carnivorous Nassu'rius reticulatus was very similar to that of the detritus­feeding Jiydrobia ventrosu, indicating that the spectra of carbohydrases cannot be predicted from information on ingested food .

5. This latter result, as well as the whole problem of a food and enzyme relation, is discussed together with the significant hydrolysis of carbohydrate which does not occur in the natural food .

Adc7WWledgemenia. Finanoial support from Statens Natur­videnskabJige ForskingsrM is gratefully acknowledged. Thanks are also due to the Copenhagen Pectin Factory and the Litex Faotory, Glostrup, for gifts of algal polysaccharides. My sincere gratitude is due to my colleague Dr. T. FENCHEL for his encouragement and interest in this work, and to Miss E. GLOB, who carefully carried out the laborious quantitative work.

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Author's address: J. HYLLEBERG KRISTENSEN Zoological Institute Laboratorium B, Ecology University of Aarhus 8000 Aarhus C Denmark

Date of final manuscript acceptance: February 4, 1972. Communicated by B. SWEDMARK, Fiskebiickskil